ABSTRACT Peripheral arterial disease (PAD) affects 8 million Americans and results in pain, gangrene, and limb amputation. Current treatments are limited. We previously demonstrated that human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) can improve blood perfusion in animal models of PAD; however, their angiogenic potential remains limited. While many single-variable (univariate) matrix studies have emphasized the importance of matrix-based cues for endothelial cell survival and function, few have focused on understanding these processes in multivariate materials, which mimic the complexity of the natural extracellular matrix (ECM). To address this limitation, we develop a combinatorial family of engineered ECMs (eECMs) with independently tunable biochemical and biomechanical cues, including stiffness and stress relaxation rate for high-throughput, matrix array studies of iPSC-EC survival and angiogenic potential. In Aim 1, we test the hypothesis that multivariate analysis will lead to the identification of optimal eECMs that enhance the regenerative capacity of iPSC-ECs and uncover previously unknown cross-talk between distinct matrix cues. Preliminary work using matrix arrays of naturally derived ECM components identified several previously unknown synergistic and antagonistic interactions between matrix cues. We build on these exciting results by creating a new array platform for combinatorial screening of modular eECMs designed for clinical translation. The eECM is an injectable hydrogel composed of recombinantly engineered matrix-mimetic proteins and polyethylene glycol crosslinked using dynamic covalent chemistry (DCC). Matrix biochemical cues are modified through protein engineering, while gel stiffness and stress relaxation rate are independently tuned through the number and kinetics of crosslinks, respectively. An in vitro array of 279 unique, combinatorial eECMs will be screened for iPSC-EC viability, phenotype, and function. Multi-factorial mathematical analyses will rank the relative importance of each eECM variable, as well as interaction effects that lead to synergistic enhancement. Results will be validated using conventional tissue culture assays. In Aim 2, we test the hypothesis that iPSC-ECs on pro-angiogenic, multivariate eECMs will have a distinctive, mechanistic signature. Integrin-mediated signaling pathways will be quantitatively assessed to correlate observed angiogenic responses to mechanistic pathways, and confirmed through gain- and loss- of-function studies. RNA sequencing will reveal new pathways and driver genes mediating the process, ultimately demonstrating a molecular signature characteristic of pro-angiogenic effects of multivariate eECMs. In Aim 3, we perform in vivo validation of the therapeutic potential of iPSC-ECs within the optimal eECM in a murine model of PAD. Controls include cells seeded in eECM with univariate cell-binding ligands or non- optimal mechanical properties, or cells delivered in saline. Cell survival will be tracked by bioluminescence imaging; laser Doppler spectroscopy and histology will determine vascular regeneration.